Activation of the chicken Ig-  locus by the collaboration of scattered regulatory regions through changes in chromatin structure

Abstract

A total of 10 B-lymphocyte-specific DNase I hypersensitive sites located in the chicken Ig-β locus were divided into four regions and combinations of deletions of these regions were carried out. A decrease in transcription of the Ig-β gene to <3% was demonstrated in cells with deletions in all four regions. The Ig-β chromatin was resistant to DNase I digestion in these cells. Thus, the collaboration is shown to convert the Ig-β chromatin from the condensed state to a relaxed state. H3 and H4 acetylation decreased to <8% but H3K4 hypermethylation was observed at the Ig-β promoter and exon 3. The collaboration of four regions had virtually no effect on CG hypomethylation in the region upstream the transcriptional start site. Accordingly, neither the DNase I general sensitive state in the Ig-β chromatin nor hyperacetylation of H3 and H4 histones in the promoter proximal region causes H3K4 di-methylation or CG hypomethylation in the promoter. From these analyses, a chromatin situation was found in which both an active state, such as enhanced H3K4 methylation, or CG hypomethylation, and an inactive state, such as DNase I resistance in the Ig-β chromatin or hypoacetylation of H3 and H4 histones in the Ig-β locus, coexist.

Transcriptional and epigenetic effects of deleting large regions, alone or in combination, from their natural context in the chicken Ig-β gene

“…S1. Southern hybridization was carried out as described previously (Shimada et al, 2006). Probe DNA was labeled using the random-priming method and [α-32 P] dCTP.…”

“…Primers for PCR analyses are described in Supplementary Table SIII and described in Itaya et al Ig-β cDNA content was normalized relative to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cDNA. Real-time PCR primers for chromatin immunoprecipitation (ChIP) assay were described previously (Shimada et al, 2006).…”

“…The state of CG methylation in the Ig-β promoter (−200 to +14) was examined using the bisulfite 0 10 20 -10 -20 kb 30 sequencing method (Clark et al, 1994) as described previously (Shimada et al, 2006).…”

“…Each smaller deletion reduced Ig-β mRNA levels in the following order IV N III N II N US1 N I Using single or combinatorial deletion of small and large deletions, the relative effects of individual deletions on Ig-β mRNA content were examined (Itaya et al, 2011;Shimada et al, 2006) (Table 1). Combinatorial deletions with IV, III, and II had decreasing effects on mRNA content; i.e., deletion IV caused the largest reduction and deletion II caused the least reduction (Shimada et al, 2006) (Table 1). The Ig-β mRNA content in triple knock-out cells, I + III+ IV Del and II+ III + IV Del cells, was 14% and 8%, respectively (Shimada et al, 2006); therefore, region II apparently enhanced Ig-β mRNA accumulation more than region I did.…”

“…Combinatorial deletions with IV, III, and II had decreasing effects on mRNA content; i.e., deletion IV caused the largest reduction and deletion II caused the least reduction (Shimada et al, 2006) (Table 1). The Ig-β mRNA content in triple knock-out cells, I + III+ IV Del and II+ III + IV Del cells, was 14% and 8%, respectively (Shimada et al, 2006); therefore, region II apparently enhanced Ig-β mRNA accumulation more than region I did. Based on these results, we concluded that the order of transcriptional activation activity was as follows: IVN III N II N I, with region IV having the greatest activity and region I having the least activity.…”